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3.5.1.1: asparaginase

This is an abbreviated version!
For detailed information about asparaginase, go to the full flat file.

Word Map on EC 3.5.1.1

Reaction

L-asparagine
+
H2O
=
L-aspartate
 +
NH3

Synonyms

14270 ASNase, alpha-asparaginase, AnsA, ansA3, AnsB, ansZ, Asn, ASNase, ASNase1, ASNase3, ASP1, ASP3, asparaginase, asparaginase II, asparagine amidohydrolase, asparginase II, ASPG, ASPG II, ASPGA1, ASPGB1, ASRGL1, At3g16150, colaspase, crasnitin, DiAsp, EcAII, EcAIII, EcaL-ASNase, ECAR-LANS, elspar, ErA, Erwinase, glutaminase-free L-asparaginase, glutamine-(asparagin-)ase, HPA, L-ASNase, L-asparaginase, L-asparaginase 2, L-asparaginase I, L-asparaginase II, L-asparaginase III, L-asparaginase type II, L-asparaginase type III, L-asparaginase-I, L-asparaginase-II, L-asparagine amidohydrolase, L-asparagine amino hydrolase, L-asparagine aminohydrolase, leunase, PfA, PhA, potassium-dependent asparaginase, potassium-independent asparaginase, potassium-independent L-asparaginase, SAMN05216263_10744, SGR ASNase, type I L-asparaginase, type II L-asparaginase, Ypa

ECTree

     3 Hydrolases
         3.5 Acting on carbon-nitrogen bonds, other than peptide bonds
             3.5.1 In linear amides
                3.5.1.1 asparaginase

Engineering

Engineering on EC 3.5.1.1 - asparaginase

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PROTEIN VARIANTS
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
F162L
-
site-directed mutagenesis of ASPGB1, mutation of Phe162 immediately preceding the variable loop in K+-dependent ASPGB1 specifically affects catalytic activity with Asn, the mutant shows an 8.4fold decrease in Vmax value with Asn, whereas the Vmax with beta-Asp-His is similar to that of the wild-type enzyme
F162W
-
site-directed mutagenesis of ASPGB1, mutation of Phe162 immediately preceding the variable loop in K+-dependent ASPGB1 specifically affects catalytic activity with Asn, the mutant shows a 4fold decrease in Vmax value with Asn, whereas the Vmax with beta-Asp-His is similar to that of the wild-type enzyme
L163F
-
site-directed mutagenesis of ASPGA1, introduction of the more bulky residue in the ASPGA1 mutant only affects Km and Vmax values with beta-Asp-His and not those with Asn, with beta-Asp-His, the Vmax value of the L163F mutant is reduced by approximately fivefold and the Km value by twofold, compared to the wild-type enzyme
N184A
-
site-directed mutagenesis of ASPGB1, the mutant shows a 3fold decrease in Vmax value with Asn as substrate
N184D
-
site-directed mutagenesis of ASPGB1, the mutant shows altered kinetics with substrate compared to the wild-type enzyme
N184Q
-
site-directed mutagenesis of ASPGB1, the mutant shows a Vmax value with Asn as substrate that is similar or slightly higher than that of the wild-type ASPGB1
R165T
-
site-directed mutagenesis of ASPGB1, the mutant shows altered kinetics with substrate compared to the wild-type enzyme
S189A
-
site-directed mutagenesis of ASPGB1, the mutant shows a 3fold decrease in Vmax value with Asn as substrate
S189C
-
site-directed mutagenesis of ASPGB1, the mutant shows altered kinetics with substrate compared to the wild-type enzyme
S189T
-
site-directed mutagenesis of ASPGB1, the mutant shows a Vmax value with Asn as substrate that is similar or slightly higher than that of the wild-type ASPGB1
T166R
-
site-directed mutagenesis of ASPGA1, introduction of the more bulky residue in the ASPGA1 mutant only affects Km and Vmax values with beta-Asp-His and not those with Asn
D289T
-
highly stabilized mutant
E260F
-
thermodynamically stabilized mutant
E292S
-
thermodynamically stabilized mutant
S180N
-
highly stabilized mutant
S180N/D289T/E260F/E292S
-
mutant exhibits a 8.1-fold increase in half-life at 65°C and a 5.56 degrees increase in melting temperature and also displays a substantial increase in the transition state energy barrier and a clear decrease in folding free energy relative to the wild-type
D289T
-
highly stabilized mutant
-
E260F
-
thermodynamically stabilized mutant
-
E292S
-
thermodynamically stabilized mutant
-
S180N
-
highly stabilized mutant
-
S180N/D289T/E260F/E292S
-
mutant exhibits a 8.1-fold increase in half-life at 65°C and a 5.56 degrees increase in melting temperature and also displays a substantial increase in the transition state energy barrier and a clear decrease in folding free energy relative to the wild-type
-
D133I
analysis of the contribution of this position on thermostability
D133L
analysis of the contribution of this position on thermostability
D133T
analysis of the contribution of this position on thermostability
D133V
analysis of the contribution of this position on thermostability
N281D
-
mutant has approximately the same specific activity as the wild-type enzyme. Mutation results in a lower glutaminase activity compared with wild-type and the N41D mutant. Mutation imparts less stability to the enzyme at elevated temperatures. The N281D mutation causes a decrease in substrate affinity as well as a decrease in the stability profile. The deamidation at the N281 site should not be a concern during processing, storage or clinical use. The deamidated variant is active and stable under normal storage conditions
N41D
-
mutant has approximately the same specific activity as the wild-type enzyme. Mutation conferrs a slight increase in kcat. Charge differences to the wild-type enzyme, at -1 per monomer or -4 per tetramer. The deamidation at the N41 site should not be a concern during processing, storage or clinical use. These deamidated variant is active and stable under normal storage conditions
N41D/N281D
-
mutant enzyme has increased specific activity. Charge differences to the wild-type enzyme, at -1 per monomer or -4 per tetramer. The N281D mutation causes a decrease in substrate affinity as well as a decrease in the stability profile. The deamidation at the N41 and N281 sites should not be a concern during processing, storage or clinical use. These deamidated variant is active and stable under normal storage conditions
N281D
-
mutant has approximately the same specific activity as the wild-type enzyme. Mutation results in a lower glutaminase activity compared with wild-type and the N41D mutant. Mutation imparts less stability to the enzyme at elevated temperatures. The N281D mutation causes a decrease in substrate affinity as well as a decrease in the stability profile. The deamidation at the N281 site should not be a concern during processing, storage or clinical use. The deamidated variant is active and stable under normal storage conditions
-
N41D
-
mutant has approximately the same specific activity as the wild-type enzyme. Mutation conferrs a slight increase in kcat. Charge differences to the wild-type enzyme, at -1 per monomer or -4 per tetramer. The deamidation at the N41 site should not be a concern during processing, storage or clinical use. These deamidated variant is active and stable under normal storage conditions
-
N41D/N281D
-
mutant enzyme has increased specific activity. Charge differences to the wild-type enzyme, at -1 per monomer or -4 per tetramer. The N281D mutation causes a decrease in substrate affinity as well as a decrease in the stability profile. The deamidation at the N41 and N281 sites should not be a concern during processing, storage or clinical use. These deamidated variant is active and stable under normal storage conditions
-
D178P
-
mutation enhances the thermostability of the enzyme without changing the activity of the enzyme and thus the therapeutical use of L-asparaginase II might be benefit from these result
D90E
active site mutation
G11V
-
518fold decrease in the ratio of turnover number to Km-value for L-aspartic acid beta-hydroxamate as substrate compared to wild-type value
G57A
-
3.8fold decrease in the ratio of turnover number to Km-value for L-aspartic acid beta-hydroxamate as substrate compared to wild-type value, 5.2fold decrease in the ratio of turnover number to Km-value for L-aspartic acid beta-hydroxamate as substrate compared to wild-type value
G57L
-
346fold decrease in the ratio of turnover number to Km-value for L-aspartic acid beta-hydroxamate as substrate compared to wild-type value
G57V
-
48.8fold decrease in the ratio of turnover number to Km-value for L-aspartic acid beta-hydroxamate as substrate compared to wild-type value, 37fold decrease in the ratio of turnover number to Km-value for L-aspartic acid beta-hydroxamate as substrate compared to wild-type value
G88A
-
8300fold decrease in the ratio of turnover number to Km-value for L-aspartic acid beta-hydroxamate as substrate compared to wild-type value
K196A/H197A
-
investigation of antigenicity, purification of mutant protein
N248A
-
5.9fold decrease in the ratio of turnover number to Km-value for L-aspartic acid beta-hydroxamate as substrate compared to wild-type value, 4657fold decrease in the ratio of turnover number to Km-value for L-aspartic acid beta-hydroxamate as substrate compared to wild-type value. Loss in transition state stabilization is 15 kJ per mol for L-glutamine, 4 kJ per mol for L-aspartic beta-hydroxamate and 7 kJ per mol for L-asparagine
N248D
-
10.18fold decrease in the ratio of turnover number to Km-value for L-aspartic acid beta-hydroxamate as substrate compared to wild-type value, 49fold decrease in the ratio of turnover number to Km-value for L-aspartic acid beta-hydroxamate as substrate compared to wild-type value. Loss in transition state stabilization is 10 kJ per mol for L-glutamine and 6 kJ per mol for L-aspartic beta-hydroxamate
N248E
-
4.4fold decrease in the ratio of turnover number to Km-value for L-aspartic acid beta-hydroxamate as substrate compared to wild-type value, 34.4fold decrease in the ratio of turnover number to Km-value for L-aspartic acid beta-hydroxamate as substrate compared to wild-type value. Loss in transition state stabilization is 9 kJ per mol for L-glutamine and 4 kJ per mol for L-aspartic beta-hydroxamate
N248G
-
7.5fold decrease in the ratio of turnover number to Km-value for L-aspartic acid beta-hydroxamate as substrate compared to wild-type value, 116fold decrease in the ratio of turnover number to Km-value for L-aspartic acid beta-hydroxamate as substrate compared to wild-type value. Loss in transition state stabilization is 12 kJ per mol for L-glutamine and 5 kJ per mol for L-aspartic beta-hydroxamate
N248Q
-
5.9fold decrease in the ratio of turnover number to Km-value for L-aspartic acid beta-hydroxamate as substrate compared to wild-type value, 6.2fold decrease in the ratio of turnover number to Km-value for L-aspartic acid beta-hydroxamate as substrate compared to wild-type value. Loss in transition state stabilization is 10 kJ per mol for L-glutamine and 4 kJ per mol for L-aspartic beta-hydroxamate
N24A
-
increase in activity compared to wild-type, a unique hydrogen bond network contributes to higher activity
N24A/R195S
-
activity similar to wild-type
N24A/Y250L
-
about 75% of wild-type activity. Mutation Y250L is an interface mutation selected to stablize the active tetramer
N24G
-
mutant has a much higher loop flexibility compared with those of wild-type and the other mutants, and a decreased catalytic activity
N24H
-
mutant displays low flexibility in the central part of the loop; the C-terminal region of the loop shows high RMSF values that are likely to cause stability problems
N24S
mutant shows completely preserved asparaginase and glutaminase activities, long-term storage stability, improved thermal parameters, and good resistance to proteases derived from leukaemia cells. The mutant displays a modification in the hydrogen bond network related to residue 24, and a general rigidification of the monomer as compared to wild-type
N24S/D281E
-
RMSF profile similar to that of WT, with a slight increase in flexibility for residues 20-24
N24T
-
increase in activity compared to wild-type. Mutant has very stable lid-loops, resulting in a tightly locked substrate molecule in the active site, stabilized for the catalytic reaction
N24T/R195S
-
about 85% of wild-type activity. Mutation R195S is an interface mutation selected to stablize the active tetramer
N24T/Y250L
-
about 70% of wild-type activity. Mutation Y250L is an interface mutation selected to stablize the active tetramer
Q59A
-
163fold decrease in the ratio of turnover number to Km-value for L-aspartic acid beta-hydroxamate as substrate compared to wild-type value, 930fold decrease in the ratio of turnover number to Km-value for L-aspartic acid beta-hydroxamate as substrate compared to wild-type value. Loss in transition state stabilization is 17 kJ per mol for L-glutamine and 13 kJ per mol for L-aspartic beta-hydroxamate
Q59E
-
15.4fold decrease in the ratio of turnover number to Km-value for L-aspartic acid beta-hydroxamate as substrate compared to wild-type value, 93fold decrease in the ratio of turnover number to Km-value for L-aspartic acid beta-hydroxamate as substrate compared to wild-type value. Loss in transition state stabilization is 7 kJ per mol for L-glutamine and 11 kJ per mol for L-aspartic beta-hydroxamate
Q59G
-
105fold decrease in the ratio of turnover number to Km-value for L-aspartic acid beta-hydroxamate as substrate compared to wild-type value, 465fold decrease in the ratio of turnover number to Km-value for L-aspartic acid beta-hydroxamate as substrate compared to wild-type value. Loss in transition state stabilization is 15 kJ per mol for L-glutamine and 12 kJ per mol for L-aspartic beta-hydroxamate
R195A/H197A
-
investigation of antigenicity, purification of mutant protein
R195A/K196A
-
investigation of antigenicity, purification of mutant protein
R195A/K196A/H197A
R240A
-
mutation increases the [S]0.5 value to 5.9 mM, presumably by reducing the affinity of the site for L-asparagine, although the enzyme retains cooperativity
S58A
-
crystallization of the mutant L-asparaginase II
T162A
-
mutation results in an active enzyme with no cooperativity
T179A
does not undergo autoprocessing
V27L
-
1.13fold increase in the ratio of turnover number to Km-value for L-aspartic acid beta-hydroxamate as substrate compared to wild-type value, 4.4fold decrease in the ratio of turnover number to Km-value for L-aspartic acid beta-hydroxamate as substrate compared to wild-type value
V27M
-
1.5fold decrease in the ratio of turnover number to Km-value for L-aspartic acid beta-hydroxamate as substrate compared to wild-type value, 11.6fold decrease in the ratio of turnover number to Km-value for L-aspartic acid beta-hydroxamate as substrate compared to wild-type value
M121C
M121C/T169M
T169M
T95D
replacement of catalytic threonine, depletes the enzyme of its catalytic activities with L-asparagine and L-glutamine
M121C
-
about 2.5fold reduced activtiy
-
M121C/T169M
-
mutant has a preserved efficiency vs L-asparagine but is completely unable to carry out L-glutamine hydrolysis. The mutant does not exert any cytotoxic effect on HL-60 cells
-
Q63E
-
mutant displays similar catalytic efficiency versus asparagine and halved glutaminase efficiency with respect to the wild type enzyme, is able to exert a cytotoxic effect comparable to, or higher than, the one of the wild type enzyme
-
T169M
-
about 4fold reduced catalytic activity
-
T16D
-
replacement of catalytic threonine, depletes the enzyme of its catalytic activities with L-asparagine and L-glutamine
-
M121C
-
mutants has nearly 2.5fold reduced catalytic activity compared to the wild type enzyme
-
M121C/T169M
-
the mutant has a preserved efficiency versus L-asparagine but is completely unable to carry out L-glutamine hydrolysis
-
T169M
-
mutants has nearly 4fold reduced catalytic activity compared to the wild type enzyme
-
T16D
-
inactive
-
T95E
-
inactive
-
T168S
the mutant shows no self-cleavage
T186V
the inactive mutant shows partial (45%) self-cleavage
T219A
the inactive mutant shows a complete, albeit slow self-cleavage
T219V
the inactive mutant displays partial (50%) self-cleavage
R206H
-
Arg206 to histidine followed by covalent coupling of mPEG-SNHS [methoxypoly(ethyene glycol) succinate N-hydroxysuccinimide ester] to the mutant enzyme results in an improved modified form of EcaL-ASNase that retains 82% of the original catalytic activity, exhibits enhanced resistance to trypsin degradation, and has higher thermal stability compared with the wild-type enzyme
V26A/E30G/D181G/V245G/G276D
-
21fold increase in catalytic efficiency, mutant shows tolerance toward wider range of pH values and higher temperatures than its wild-type counterpart
V26A/E30G/K122N/G276D
-
20fold increase in catalytic efficiency
V26A/E30G/D181G/V245G/G276D
-
21fold increase in catalytic efficiency, mutant shows tolerance toward wider range of pH values and higher temperatures than its wild-type counterpart
-
V26A/E30G/K122N/G276D
-
20fold increase in catalytic efficiency
-
K274E
site-directed mutagenesis, the active site mutant shows improved enzymatic properties at physiological conditions compared to the wild-type. The mutant is thermodynamically stable and resistant to proteolytic digestion, displays no glutaminase activity, and shows increased and more significant killing of human cell lines HL60, MCF7, and K562 as compared to the Escherichia coli L-asparaginase
T53Q
site-directed mutagenesis, the active site mutant shows improved enzymatic properties at physiological conditions compared to the wild-type. The mutant is thermodynamically stable and resistant to proteolytic digestion, displays no glutaminase activity, and shows increased and more significant killing of human cell lines HL60, MCF7, and K562 as compared to the Escherichia coli L-asparaginase
T53Q/K274E
site-directed mutagenesis, the active site mutant shows improved enzymatic properties at physiological conditions compared to the wild-type. The mutant is thermodynamically stable and resistant to proteolytic digestion, displays no glutaminase activity, and shows increased and more significant killing of human cell lines HL60, MCF7, and K562 as compared to the Escherichia coli L-asparaginase
W301F
-
mutation has no effect on secondary and tertiary structure of the protein. Initiation of unfolding transition of the W301F protein happens at a higher GdnCl concentration compared to W60F, indicated that the N-domain is more stable compared to the C-domain
W60F
-
mutation has no effect on secondary and tertiary structure of the protein. Initiation of unfolding transition of the W301F protein happens at a higher GdnCl concentration compared to W60F, indicated that the N-domain is more stable compared to the C-domain
K215A
-
99.9% loss of activity
T141A
-
99.9% loss of activity
T64A
-
99.9% loss of activity
Y78A
-
99.9% loss of activity
K215A
-
99.9% loss of activity
-
T141A
-
99.9% loss of activity
-
T64A
-
99.9% loss of activity
-
Y78A
-
99.9% loss of activity
-
S121P
additional information